Efficacy and Safety of Dual Paclitaxel and Sirolimus Nanoparticle-Coated Balloon

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In vitro cell culture studies
In the initial set of experiments, keeping the SRL concentration constant in solution (100 or 1000 ng/ml), the cells were treated with different doses of PTX in solution to determine the most effective synergistic drug combination.The subsequent experiment was carried out as above with the encapsulated PTX and SRL and finally with co-encapsulated PTX and SRL nanoparticle formulation.The inhibition of cell proliferation was determined using the CyQUANT™ NF Cell Proliferation Assay kit (Life Technologies, Carlsbad, CA).The IC50 for each treatment was calculated using the non-linear curve fit (logistic) using OriginPro 8 (OriginLab Corp., Northampton, MA).The above series of experiments determined that the 1:9 w/w ratio of PTX: SRL is highly synergistic; hence, the above ratio of the two drugs co-encapsulated in NPs was further investigated, particularly to understand the mechanism of inhibition of cell proliferation.It was demonstrated that encapsulation enhances the antiproliferative effect of the combination treatment as compared to drugs in solution in the same combination ratio.

Analyzing dead/live cells and BrdU positive proliferating cells in cell culture experiment
NPs without any encapsulated API acted as a control group.The IC50 for each treatment was determined in HVSM cells (SRL=3,100 ng/mL, PTX = 45 ng/mL, and combination = 225 ng/mL).
Four hours prior to 3 day incubation period, the cells were imaged using an EVOS XL Core phase contrast microscope (Advanced Microscopy Group, Bothell, WA).From these images, total cells and apoptotic/dead cells were counted from ten random fields out of three replicate wells, and the results were plotted as a percentage of apoptotic cells with respect to the total number of cells.
Immediately after imaging, the cells were incubated with BrdU (5-bromo-2'-deoxyuridine), Thymidine analog (Cat# ab142567, Abcam, Cambridge, MA) dissolved in cell culture media at 10 µM concentration for four hours to determine proliferating cells.The cell culture medium was removed from the well, and the cells were fixed with 4% paraformaldehyde.The cells following immunocytochemical staining with eBioscience BrdU kit (Cat# 8800-6599, ThermoFisher Scientific) and counterstaining with hematoxylin were imaged using Aperio AT2 DX microscope (Leica Biosystems, Wetzler, Germany).Anti-BrdU stained proliferative positive cells were identified and counted manually from ten randomly selected areas of the images using Aperio ImageScope software (Leica Biosystems)

Tissue processing and histology preparation in rabbit iliac model experiment
Immediately after euthanasia, the treated iliofemoral arteries were carefully dissected free from surrounding tissue and flushed with 50-100 ml of heparinized lactate ringers to remove residual blood.All the tissue was treated in stages with alcohol and xylene.After dehydration, each sample was further sectioned into 3-4 mm intervals and sequentially placed in a block, keeping the longitudinal orientation.Tissue samples were cut into 5-6 µm sections with a rotary microtome, mounted on charged slides, and stained with hematoxylin and eosin (H&E) and modified Movat Pentachrome (MP) stains.For the immunohistochemical evaluation, the primary antibody for BrdU (cat#MO744, DAKO/Agilent, Santa Clara, CA) was used at a concentration of 1:200.The effect of BrdU was confirmed by sections of the small intestine of each animal (internal positive control).

Morphometry and histology analysis in rabbit iliac model experiment
Quantitative assessments were performed to determine the morphologic effects of treatment.MPstained histological sections were analyzed using digital planimetry with a microscope system calibrated to perform the measurements [NIST Trackable Calibrated Microscope System (IP Lab Software, Rockville, MD)].Subsequently, to assess arterial injury and healing, histologic parameters were examined for endothelial loss, platelets/fibrin, surface and medial, medial extracellular matrix (ECM)/collagen, calcification, intimal/medial and adventitial inflammation, medial and adventitial red blood cells (bleeding), medial SMC loss and medial injury.

Coronary artery catheterization and intervention in swine heart model
At the initial procedure on day 0, angiography was performed with contrast medium after the administration of nitroglycerin (100 µg), then all the devices were delivered over a 0.014-inch guidewire to the target vessels.Endothelial denudation was performed at targeted sites using a plain uncoated balloon at the selected sites prior to treatment with DCB.Balloon expansion pressure for the denudation was based on the online Quantitative coronary angiography (QCA) diameter and the provided balloon compliance chart.Dual-API DCB or PTX-DCB 3.0 x 20 mm were then deployed over a guidewire at the target site in the LAD or LCX, and POBA 3.0 x 20 mm was deployed in the RCA.Each balloon was inflated for 60 seconds with a target balloon-tovessel ratio of at least 10% based on a compliance chart that achieved a range of 0.99:1 to 1.3:1.
Overall, both the LAD and LCX of each animal were treated with either Dual-API DCB or PTX-DCB, and the RCA of each animal was treated with POBA.
Termination of swine model experiment excised, flushed with 500-1000 mL of heparinized lactate Ringer's solution under 80-100 mmHg pressure to remove residual blood, and gravity perfusion fixed in 10% neutral buffered formalin (NBF) for 20 minutes.The heart samples were immersion-fixed overnight in 10% NBF and then transported to CVPath Institute for histological processing and analysis.

Table 3 . Percentage of cells in different phases of cell-cycle
MYO 1 and MYO 2 are two areas near the treated coronary artery.LAD= left anterior descending artery, LCX= left circumflex artery, RCA = right coronary artery, RV = right ventricle Supplemental

Table 4 .
Angiographic measurements at baseline and 28 days follow-up in porcine coronary model.

Table 5 .
Vessel morphometry and histologic results at 28 days in porcine coronary model

Table 6 .
Number of assessed sections of downstream myocardium in each animal